The invention relates generally to polypeptides and nucleic acids and more particularly to polypeptides that interact with the MDM2 polypeptide, and nucleic acids encoding these MDM2-interacting polypeptides.
The gene MDM2 has been implicated in a variety of cellular processes. In addition, altered expression of MDM2 has been associated with several disease states, including cancer. For example, the MDM2 gene has been shown to be abnormally up-regulated in human tumors and tumor cell lines. In addition, amplification of MDM2 genes has been reported in a variety of cancers, e.g., human sarcoma, glioma, squamous cell carcinoma, breast cancer, astrocytoma, leukemia and lymphoma. These results indicate that the MDM2 protein plays a role in human carcinogenesis.
MDM2 has been reported to interact with, i.e., bind to other proteins. Some of these proteins have themselves been associated with tumorigenesis. For example, MDM2 has been reported to form a complex with the p53 tumor suppressor and to block the growth suppressive functions of p53. In addition, overexpression of MDM2 has been shown to block the transactivation, cell cycle arrest (S/G2 phase) and apoptotic functions of p53.
MDM2 has also been shown to interact with the retinoblastoma tumor suppressor protein pRb, and the E2F-1 and DP1 transcription factors. Through MDM2""s activation of E2F and DP1, MDM2 is thought to stimulate a G1 to S transition in the cell cycle. In addition, MDM2 has also been reported to interact with Numb, a protein involved in the determination of cell fate.
The invention is based in part on the discovery of a novel polypeptide, named MDM Interacting Protein, or xe2x80x9cMDMIPxe2x80x9d, based on its ability to bind to the MDM2 polypeptide.
In one aspect, the invention includes a purified complex that includes a polypeptide which includes an MDMIP binding domain (or region) of an MDM2 polypeptide and a polypeptide which includes an MDM2 binding domain (or region) of an MDMIP polypeptide. The complex can thus include, e.g, an MDM2 polypeptide and an MDMIP polypeptide. In various embodiments, the MDM2 polypeptide, the MDMIP polypeptide, or both polypeptides within the complex, are human polypeptides.
In some embodiments, the MDM2 binding domain of the MDM2 polypeptide includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the amino acid sequence of a region of polypeptide that includes the amino acid sequence of SEQ ID NO:2.
In some embodiments, the MDMIP binding domain of the MDM2 polypeptide includes an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the amino acid sequence of a region of polypeptide that includes the amino acid sequence encoded by the nucleic acid sequence having GenBank accession number M92424. In some embodiments, the MDMIP binding domain includes the amino acid sequence of SEQ ID NO:4, e.g., it can include the amino acid sequence of the MDM2 polypeptide encoded by the nucleic acid sequence of M9424 (SEQ ID NO:6). Thus, in some embodiments, the MDMIP binding domain of the MDM2 polypeptide is present in a polypeptide which includes the amino acid sequence of an MDM2 polypeptide, e.g., a polypeptide including at least that portion of the amino acid sequence of SEQ ID NO:4 that is sufficient to bind to an MDMIP polypeptide.
If desired, one or more of the polypeptides in the MDM2-MDMIP complex, e.g., the MDM2-binding domain of the MDMIP polypeptide, the MDMIP-binding domain of the MDM2 polypeptide, or both is labeled.
In other embodiments, the complex includes a fragment of an MDMIP polypeptide that includes an MDM2-binding domain, a fragment of an MDM2 polypeptide that includes an MDMIP-binding domain, or both fragments.
Another aspect of the invention includes a chimeric polypeptide that includes a region of an MDMIP polypeptide covalently linked, e.g., via a peptide bond, to a region of an MDM2 polypeptide. Preferably, the chimeric peptide includes 6, 8, 10, 12, 14, or 16 or more amino acids of an MDM2 polypeptide covalently linked to 6, 8, 10, 12, 14, 16 or more amino acids of an MDMIP polypeptide.
In some embodiments, the amino acids of the MDMIP-derived polypeptide in the chimeric polypeptide include an MDM2 binding domain. For example, the MDMIP-derived polypeptide can include a polypeptide which binds to MDM2 and, optionally, includes at least a portion of an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the amino acid sequence of SEQ ID NO:2.
Similarly, in some embodiments, the amino acids of the MDM2-derived polypeptide in the chimeric polypeptide include an MDMIP-binding domain. Thus, the MDM2 polypeptide can include a polypeptide which binds to MDMIP and, optionally, includes at least a portion of an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, or 99% or more identical to the amino acid sequence of SEQ ID NO:4, i.e., a polypeptide having the amino acid sequence encoded by nucleic acids 312-963 of the nucleic acid sequence corresponding to GenBank accession number M92424.
In a further aspect, the invention includes an isolated MDMIP nucleic acid. In some embodiments, the MDMIP nucleic acid encodes an MDMIP polypeptide, e.g., the nucleic acid may include a nucleic acid that encodes a polypeptide at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or even 99% identical to a polypeptide which includes the amino acid sequence of SEQ ID NO:2.
The MDMIP nucleic acid may alternatively, or in addition, include a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or even 99% identical to a nucleic acid which includes nucleic acids 1-222 of FIG. 1 (SEQ ID NO:1).
Also provided by the invention is a vector including an MDMIP nucleic acid, as well as a cell that includes an MDMIP nucleic acid. The MDMIP nucleic acid in the cell may be present in a vector, i.e., the invention includes a cell that contains a vector in which an MDMIP nucleic acid is present.
Another aspect of the invention includes a purified MDMIP polypeptide with an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even 99% or more identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the MDMIP polypeptide binds to an MDM2 polypeptide.
In a further aspect, the invention includes an isolated MDM2-derived nucleic acid fragment. The MDM2-derived nucleic acid is less than the full length of a full-length MDM2 nucleic acid, i.e., the nucleic acid includes less than the full length reading frame of a nucleic acid encoding a full length MDM2 polypeptide. In some embodiments, the MDM2-derived nucleic acid encodes an MDM2polypeptide fragment that is less than 491 amino acids, e.g, it is less than 450, 400, 350, 300, 250, 200, 150, 100, or 50 amino acids, and, optionally, binds an MDMIP polypeptide, e.g., a polypeptide having the amino acid sequence of SEQ ID NO:2. Alternatively, or in addition, the MDM2-derived nucleic includes a nucleic acid that encodes a polypeptide at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or even 99% identical to a polypeptide which includes the amino acid sequence of SEQ ID NO:4, and which is less than 491 amino acids in length and is greater than 4 amino acids in length. Preferably, the MDMIP-binding polypeptide is greater than 5, 6, 7, 8, 9, or 10 amino acids in length and is less than 25, 50, 75, 100, 150, 200, 250, 300, 350, 400 or 450 amino acids in length.
The MDM2-derived nucleic acid may alternatively, or in addition, include a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or even 99% identical to a nucleic acid which includes the nucleic acid sequence of SEQ ID NO:3.
Also provided by the invention is a vector including the MDM2-derived nucleic acid, as well as a cell that includes an MDM2-derived nucleic acid. The MDM2-derived nucleic acid in the cell may be present in a vector, i.e., the invention includes a cell that contains a vector in which an MDM2-derived nucleic acid is present.
Another aspect of the invention includes a purified MDM2 polypeptide with an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or even 99% or more identical to a polypeptide that includes the amino acid sequence of SEQ ID NO:4.
Also provided by the invention are antibodies (e.g., polyclonal or monoclonal antibodies) that bind to the MDMIP-MDM2 complexes described herein. For example, the invention includes an antibody which specifically binds to a complex that includes a polypeptide that has an MDMIP binding domain of an MDM2 polypeptide and a polypeptide that includes an MDM2 binding domain of an MDMIP polypeptide. In some embodiments, the antibody binds with higher affinity to a complex that includes an MDMIP binding domain of an MDM2 polypeptide and an MDM2 binding domain of an MDMIP polypeptide as compared to the binding of the antibody to an isolated MDM2 polypeptide or an isolated MDMIP polypeptide.
The invention also includes antibodies (e.g, polyclonal or monoclonal antibodies) that bind to an MDMIP polypeptide, or to an MDMIP-binding fragment of an MDM2 polypeptide. These antibodies, in various embodiments, may optionally bind to the herein described MDM2-MDMIP complexes as well.
In a further aspect, the invention features a pharmaceutical composition that includes one or more of an MDMIP polypeptide, and MDMIP nucleic acid, a chimeric polypeptide containing sequences derived from an MDM2 polypeptide and sequences derived from an MDMIP polypeptide, and a complex of an MDM2-binding domain of an MDMIP polypeptide and an MDM2-binding domain of an MDMIP polypeptide. The pharmaceutical composition may alternatively, or additionally, contain an antibody to any of these polypeptides, nucleic acids or complexes.
Also included in the invention is a kit containing reagents that detect one or more of an MDMIP polypeptide, and MDMIP nucleic acid, a chimeric polypeptide containing sequences derived from an MDM2 polypeptide and sequences derived from an MDMIP polypeptide, and a complex of an MDM2-binding domain of an MDMIP polypeptide and an MDM2-binding domain of an MDMIP polypeptide. In some embodiments, the reagents can include one or more of an antibody to an MDM2 polypeptide, an antibody to and MDMIP polypeptide, and/or an antibody to a complex of an MDM2-binding domain of an MDMIP polypeptide and an MDM2-binding domain of an MDMIP polypeptide. The reagent may in addition include the MDMIP polypeptide for detecting the presence of MDM2 in a sample, an MDM2 protein for detecting the presence of MDMIP in a sample.
The invention also includes methods of identifying a MDM2-MDMIP complex, or various components of an MDM2-MDMIP complex, in a biological sample. For example, the invention includes a method of identifying a MDM2 sample in a biological sample by contacting the biological sample with an MDMIP polypeptide under conditions allowing for the formation of MDM2-MDMIP complexes. Levels of the MDM2-MDMIP complex, if present, are then determined, thus allowing for determination of levels of MDM2 polypeptide in the cell.
In some embodiments, MDM2 polypeptides are detected using a labeled MDMIP polypeptide. Detection of labeled MDMIP-MDM2 complexes indicates the presence of a MDM2 polypeptide in the sample.
The invention also features a method of purifying an MDM2 polypeptide from a biological sample by contacting the biological sample with a polypeptide that includes an MDM2-binding region of an MDMIP polypeptide.
Also included in the invention is a method of identifying an agent which modulates MDM2 or MDMIP activity. The method includes contacting a test agent with an MDM-MDMIP complex and measuring binding of the agent to the complex. Binding of the agent to the complex indicates the agent modulates MDM2 or MDMIP polypeptide activity.
In another aspect, the invention includes a method of identifying an agent which modulates MDMIP activity. In this method an MDMIP polypeptide is mixed with a test agent and binding of the agent to the complex is measured. Binding of the agent to the complex indicates the agent modulates MDMIP polypeptide activity.
The invention also includes a method of determining whether a test subject has a disorder associated with aberrant expression of MDM2 protein. The method includes providing a biological sample from the subject, measuring the level of the complex in the subject; and comparing the level of complex in the sample to the level of the complex in a reference sample whose MDM2 expression state is known. Comparing the relative levels of the MDM2 protein in the two samples makes it possible to determine whether the subject has a disorder associated with aberrant expression of an MDM2 protein.
Yet another aspect of the invention is a method of treating or preventing a disease or disorder involving aberrant levels of a complex of a MDM2 protein and a MDMIP. The method includes administering to a subject in which such treatment or prevention is desired a therapeutically-effective amount of a molecule or molecules which are capable of modulating the function of the complex.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.